Local Peripheral Effects of Β-Caryophyllene Through CB2 Receptors in Neuropathic Pain in Mice

Home , Caryophyllene

Pharmacology & Pharmacy, 2012, 3, 397-403 1 http://dx.doi.org/10.4236/pp.2012.34053 Published Online October 2012 (http://www.SciRP.org/journal/pp)

Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice

Hikari Kuwahata1,2, Soh Katsuyama2,3, Takaaki Komatsu2, Hitoshi Nakamura3, Maria Tiziana Corasaniti4, Giacinto Bagetta5, Shinobu Sakurada6, Tsukasa Sakurada2*, Kazuo Takahama1

1Department of Environmental and Molecular Health and Sciences, Graduated School of Pharmaceutical Sciences, Kumamoto Uni- versity, Kumamoto, Japan; 2Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan; 3Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, Sendai, Japan; 4Department of Pharmacobiological Sciences, Univer- sity of Magna Gracia of Catanzaro, Catanzaro, Italy; 5Department of Pharmacobiology, University Consortium for Adaptive Disor- ders and Headach (UCADH), Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Arcavacata di Rende, Italy; 6Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan. Email: *[email protected]

Received July 13th, 2012; revised August 15th, 2012; accepted September 10th, 2012

ABSTRACT β-Caryophyllene (BCP) is known as a common constitute of the essential oils of numerous food plants and primary component in Cannabis. In this study, we investigated the effect of local intraplantar (i.pl.) injection of BCP on mechanical hypersensitivity induced by partial sciatic nerve ligation (PSNL) in mice. Relative to sham operation controls, mice with the PSNL displayed a maximum level of hyperresponsiveness to von Frey metallic filament on post-operative day 7. PSNL-induced allodynia was seen in the ipsilateral side of nerve ligation, but not in the contralateral side. The i.pl. injection of BCP into the ipsilateral hindpaw to PSNL attenuated mechanical allodynia in a dose-dependent manner. BCP injection into the contralateral hindpaw did not produce anti-allodynic effects, suggesting a local peripheral anti-allodynic effect of BCP. Anti-allodynic effects induced by i.pl. injection of BCP were prevented by pretreatment with the cannabinoid (CB)2 receptor antagonist AM630, but not by the CB1 receptor antagonist AM251. These data suggest that i.pl. injection of BCP could produce anti-allodynia by activating peripheral CB2 receptors, but not CB1 receptors in a mouse model of neuropathic pain. Taken together, these results suggest that peripheral CB2 receptors may contribute to the effectiveness of BCP in the treatment of neuropathic pain disorders.

Keywords: β-Caryophyllene (BCP); Neuropathic Pain; Partial Sciatic Nerve Ligation (PSNL); Peripheral Cannabinoid (CB) Receptor

1. Introduction staglandin E1-induced edema in rats as well as anti-arthritic activity [8-10]. Oral administration of BCP sig- Plants are used for various purposes including their cos- nificantly reduced the inflammation of colon [11], the metic, nutritive, and biomedical properties. Plant essen- carrageenan-induced inflammatory response in wild-type tial oils are typically composed of volatile aromatic ter- mice but not in mice lacking cannabinoid (CB) receptors penes and phenylpropanoids. The natural sesquiterpene 2 [12]. However, the antinociceptive efficacy of intraplan- β-caryophyllene (BCP) is found in many essential oils of tar (i.pl.) BCP on partial sciatic nerve ligation (PSNL)- different spice and food plants, such as clove, oregano, induced mechanical allodynia in mice is unknown. Can- thyme, black pepper and cinnamon [1,2], all of which nabinoid CB receptors, CB and CB receptors, at the have been used as natural remedies and also as fra- 1 2 peripheral and central sites have been proposed to medi- grances. This compound is also known to be anti-micro- ate the CB-induced antinociceptive effects [13-17]. CB bial [3], anti-oxidant [3,4], and anti-carcinogenic [5] and 2 receptors are not found in the central nervous system to possess skin penetration-enhancing properties [6]. (CNS), but are predominantly expressed in immune cells, Moreover, BCP is also a major component in the essen- their roles including the modulation of cytokine release tial oil of Cannabis sativa L [7]. BCP showed marked and immune cell migration [18,19]. CB receptor selec- anti-inflammatory activity against carrageenan- and pro- 2 tive agonists produced peripheral antinociception [15, *Corresponding author. 20,21], but do not cause the effects of CNS [15,22], sug-

gesting that selective activation of CB2 receptors may tion of motor disorders or change in weight. Testing achieve the goal of peripheral pain relief without CNS procedures were conducted on day 7 after PSNL, except effects. Therefore, in our study we used local injections for the time-course experiment of PSNL-induced allody- to the injured paw in order to exclude the role of central nia. effects, and validate the role of peripheral CB receptors in neuropathic pain. Peripheral nerve damage can result 2.2. Mechanical Threshold in long-lasting anomalous pain conditions referred to as Behavioral testing was conducted from 10:00 to 16:00 in neuropathic pain. These abnormal pain states are often a quiet room. Each animal received drugs only once and manifested by an increased sensitivity to nociceptive was used in only one experiment. The mice were stimuli, termed hyperalgesia, as well as the perception of weighed and placed individually in a Plexiglas chamber typical innocuous stimuli being painful, a state referred (11.0 × 17.0 × 14.0 cm, wire mesh floor) and allowed to to as allodynia [23]. CB2 receptor selective agonists have acclimatize for at least 1 hour. The threshold for nocicep- been also known to produce antinociception without tive responsiveness to mechanical stimuli applied to the overt behavioral effects in neuropathic pain [20,22,24, hindpaw was assessed using an electronic version of the 25]. The aims of this work was 1) to investigate whether von Frey test (dynamic plantar anesthesiometer, model i.pl. injection of BCP would produce antinociception in 37400; Ugo Basile, Milan, Italy). The servo-controlled PSNL-induced mechanical allodynia model in mice; and mechanical stimulus (a pointed metallic filament) was 2) to determine a possible role of peripheral CB recap- applied to the plantar surface, which exerted a progres- tors in BCP-induced anti-allodynic effects. sively increasing punctate pressure, reaching up to 3.0 g within 5.0 s. The pressure evoking a clear voluntary hind- 2. Materials and Methods paw withdrawal response (usually close to 3.0 g) was 2.1. Animals and Neuropathic Surgery recorded automatically and taken as the mechanical nociceptive thereshold index. Male mice of ddY strain weighing 22 - 24 g were pur- chased from Kyudo Industries, Kumamoto, Japan. They 2.3. Drugs were housed in cages of 15 - 20 animals matched for weight and placed in a colony room. Animals were al- β-Caryophyllene(trans-(1R,9S)-8-Methylene-4,11,11-tri- lowed free access to standard food (Clea Japan, Inc., methylbicyclo [7.2.0] undec-4-ene; BCP) (Sigma, St. Osaka, Japan) and tap water in an air-conditioned room Louis, MO, USA) diluted in jojoba wax (Simmondsia under a constant 12:12 h light/dark cycle (light on 08:00 chinensis) (KSA International, Co. Ltd., Kanagawa, Ja- h) at a room temperature of 22˚C - 24˚C and 50% - 60% pan), was injected to the plantar surface of the hindpaw relative humidity. All experiments followed the Guide- in mice. N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlo- lines on Ethical Standards for Investigation of Experi- rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) mental Pain in Animals [26]. Additionally, the study was and 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-in- approved by the Committee of Animal Experiments in dol-3-yl] (4-methoxyphenyl) methanone (AM630) (Toc- Daiichi College of Pharmaceutical Sciences. ris Cookson, Bristol, UK), dissolved in physiological Partial ligation of the sciatic nerve of mice was per- saline was administered i.pl. or subcutaneous (s.c.) 30 formed under pentobarbital anesthesia (50 mg/kg, i.p.) min before BCP. I.pl. and s.c. injections were given in a following the methods Malmberg and Basbaum [27]. volume of 20 μL/site and 0.1 mL/10 g of body weight, Briefly, the common sciatic nerve of the right hand leg of respectively. mice was exposed at high thigh level through a small incision and dorsal 13 to 12 of the nerve thickness 2.4. Analyses of Data was tightly ligated with a silk suture. The wound was All data are expressed as means ± SEM. Statistical dif- closed with a single muscle suture, and antibiotic powder ferences between groups were assessed with a two-way was dusted over the wound area following surgery. For ANOVA followed by Bonferroni’s test. The 5% (P < sham surgery, the sciatic nerve was exposed as described 0.05) level of statistical significance was set in all ex- above, but no contact was made with the nerve. To periments. minimize differences in technique, all operations were done by the same person. Immediately following surgery, 3. Results the animals were kept in a soft bag cage with some food 3.1. The Effects of BCP on Mechanical Allodynia inside so that they could feed themselves without having difficulty in standing. The wound healed within 1 to 2 The responsiveness to mechanical stimuli was deter- days, and the mice behaved normally. The behavior of mined on days 1 - 35 after PSNL. Compared with the the mice was monitored carefully for any visual indica- sham-operated mice, PSNL resulted in mechanical allo-

Copyright © 2012 SciRes. PP Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice 399 dynia demonstrated by significantly lower threshold to BCP, whereas AM251 (12.0 μg/paw) gave no effect von Frey filaments from day 7 to day 28 post-PSNL (Figure 4). AM251and AM630 used in this experiment (Figure 1). The maximal decrease in withdrawal thresh- alone did not induce a significant effect as compared to old after PSNL was observed on day 7. On day 7 post- saline controls (data not shown). PSNL, i.pl. injection of BCP (4.5, 9.0 and 18.0 μg/paw) dose-dependently increased the mechanical response 4. Discussion threshold on the nerve injured side. The effect of BCP The present study demonstrates that i.pl. injection of reached a maximum at 5 min and lasted for 15 min on BCP reversed mechanical allodynia in a mouse PSNL nerve-ligated side. Jojoba wax control, injected into the neuropathic pain model. The anti-allodynia induced by nerve-ligated side, did not affect the mechanical thresh- BCP was inhibited by pretreatment with AM630, a selec- olds. To ensure that the effects of i.pl. injections of BCP tive CB receptor antagonist, while pretreatment with were local and not due to systemic diffusion, BCP (18.0 2 AM251, a selective CB receptor antagonist, did not μg/paw) was injected into the hindpaw contralateral. 1 change the effects of BCP. These results suggest that Mechanical threshold following i.pl. injection of BCP in the contralateral hindpaw was not statistically different BCP, injected into the plantar surface of the hindpaw, when compared with the jojoba wax control group in PSNL mice (Figure 2).

3.2. Peripheral CB2 Receptor Mediates the Effects of BCP To determine if anti-allodynic effect of BCP was mediated by peripheral CB systems, animals were pretreated sys- temically with AM251 (3.0 mg/kg, s.c.), a selective CB1 receptor antagonist and AM630 (1.0 mg/kg, s.c.), a selective CB2 receptor antagonist, 30 min before i.pl. injection of BCP (18.0 μg/paw). The selective CB2 receptor antagonist, AM630 significantly reversed the inhibitory effects of BCP on the PSNL-induced mechanical allodynia, whereas the selective CB1 receptor antagonist, Figure 1. The time course of mechanical withdrawal thresh- AM251 gave no effect (Figure 3). In further experiments, olds after partial sciatic nerve ligation in the von Frey test. AM251 and AM630 were pretreated directly into the Mechanical withdrawal thresholds of the injured and sham- operated right hindpaws are expressed as grams and data same site on the hindpaw in prior to i.pl. injection of represent the mean ± SEM of 10 mice in each group. Aster- BCP. I.pl. pretreatment with AM630 (4.0 μg/paw) could isks indicate significant decreases in thresholds compared to also antagonize significantly anti-allodynic effects of the sham-operated mice. *P < 0.05, **P < 0.01.

Figure 2. Intraplantar injection of BCP reduced partial sciatic nerve ligation (PSNL)-induced mechanical allodynia in mice. The withdrawal threshold was determined in PSNL and sham mice (hindpaw ipsilateral side and contralateral side) before and 5, 10, 15, 30, 60 and 90 min after administration. Each point represents the mean ± SEM for groups of 10 mice. **P < 0.01, *P < 0.05 when compared to jojoba wax-control.

Figure 3. Antagonism induced by s.c. injection of the selective CB2 receptor antagonist, AM630 and the selective CB1 receptor antagonist, AM251 on the anti-allodynia produced by i.pl. injection of BCP in the PSNL model. Each point represents the mean ± SEM for groups of 10 mice. **P < 0.01 when compared to saline (s.c.) + jojoba wax-control. ##P < 0.01 when compared to s.c. saline plus BCP (18.0 μg/paw).

Figure 4. Antagonism induced by local injection of the selective CB2 receptor antagonist, AM630 and the selective CB1 receptor antagonist, AM251 on the anti-allodynia produced by i.pl. injection of BCP in the PSNL model. Each point represents the mean ± SEM for groups of 10 mice. **P < 0.01 when compared to saline (s.c.) jojoba wax-control. ##P < 0.01 when compared to i.pl. saline + BCP (18.0 μg/paw). produces its anti-allodynic effects by activating local pe- eral to the injured side could protect the mice from me- ripheral CB2 receptors. chanical allodynia induced by PSNL. Local injection of Injury to the sciatic nerve in mice as well as rats pro- BCP into the contralateral hindpaw gave no effects on duces a prolonged mechanical allodynia and thermal hy- mechanical allodynia, strongly supporting a local effect peralgesia [27,28]. This experimental model has estab- of BCP on peripheral cutaneous nociceptors. It seems lished as a reliable and objective method to access neu- that i.pl. treatment with BCP is beneficial as BCP is ropathic pain. We confirmed that PSNL produced a rapid anti-allodynic in neuropathic pain and have limited CNS onset and prolonged mechanical allodynia in mice. The penetration to produce the unwanted CNS effects. It mechanical allodynia developed in 1 day and reached a should be noted that locally applied BCP failed to pro- maximum in 7 days in mice with PSNL. PSNL-induced duce anti-allodynic effects in sham-operated mice at a mechanical allodynia was dose-dependently reversed by dose of 18.0 μg/paw. At this dose, BCP attenuated sig- local (plantar surface of the hindpaw) injection of BCP nificantly mechanical hypersensitivity in neuropathic on day 7 post-PSNL. This result suggests that i.pl. inject- mice. The employment of CB receptor-selective antago- tion of BCP is an effective post-injury treatment in neu- nists allows for the involvement of CB receptor subtypes ropathic pain conditions. in BCP-induced anti-allodynia. Locally applied BCP (9.0 In this study, injection of BCP into the hindpaw ipilat- and 18.0 μg/paw) significantly reduced the PSNL-in-

Copyright © 2012 SciRes. PP Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice 401 duced neuropathic pain in mice. The effects of BCP were paw [34]. Therefore, it seems that activation of periph- inhibited by pretreatment with the selective CB2 receptor eral CB2 receptors might decrease the sensitivity of pri- antagonist, AM630 (1.0 mg/kg, s.c. and 4.0 μg, i.pl.) but mary afferent neurons by inhibiting the release of sensi- not by pretreatment with the selective CB1 receptor an- tizing substances from neighboring mast and immune tagonist, AM251 (3.0 mg/kg, s.c. and 12.0 μg, i.pl.). The cells. Moreover, there is evidence for the presence of doses AM630 (1 mg/kg) and AM251 (3 mg/kg) used in CB2 receptors on peripheral nerve terminals [42,43]. In- the present experiment have been previously shown to deed, Walczak et al. (2005) have shown that the saphe- inhibit the effect of WIN 55,212-2, a CB receptor agonist nous partial ligation-induced neuropathic pain model in- in vivo [18,29]. In binding assays, AM630 is CB2 selec- creases the expression of CB2 receptors in the paw skin tive with 165-fold lower Ki value in CB2 transfected [44]. Another action mechanism of BCP is that i.pl. BCP CHO cell membranes than in membranes of CB1 trans- may stimulate CB2 receptors and inhibit the responsive- fected cells [30], and AM251 is a high degree selectivity ness of primary afferent neurons by stimulating local (306-fold) for CB1 receptors [31]. Therefore, the data release of β-endorphin, an endogenous opioid peptide, suggests that the anti-hyperalgesic effects of BCP in from keratinocytes, which are very abundant in skin and neuropathic mice are mediated through activation of pe- have been reported to express CB2 receptors [35]. How- ripheral CB2 receptors. In line with our study, systemic ever, further studies are required to explain the specific or local administration of CB2 receptor selective agonists mechanisms underlying the observed BCP effect. have been shown to produce antinociception without overt behavioral effects in several pain models [15,20,32,33]. 5. Conclusion Besides peripherally mediated anti-allodynic effect, In conclusion, i.pl. injection of BCP reduced the me- peripheral administration of BCP or its containing essen- chanical allodynia by the PSNL model in mice. The anti- tial oil also indicates anti-inflammatory and anti-arthritic allodynic effects of BCP was antagonized by s.c. and i.pl. potential [8-11,34]. Moreover, oral administration of pretreatment with the selective CB receptor antagonist, BCP inhibits potent anti-inflammatory effects in wild- 2 AM630, but not by the selective CB receptor antagonist, type mice but not in mice lacking CB receptors [12]. 1 2 AM251. The results suggest that a local effect of BCP on This is consistent with our present study, suggesting the cutaneous nociceptors is mediated through CB receptors. involvement of specific CB receptor subtype, a CB re- 2 2 BCP is predicted to be effective in treating allodynia ceptor, in BCP-induced peripheral anti-allodynia. Inter- without the central side effects of cannabinoids-based estingly, the essential oil component (E)-BCP selectively drugs retaining activity at the CB1 receptor. binds to the CB2 receptor, leading to the cellular activation as a CB2 agonist and anti-inflammatory effects [12]. Thus, the anti-allodynic effect of BCP points to the pe- REFERENCES ripheral CB2 receptor as an interesting target in searching [1] A. Di Sotto, M. G. Evandri and G. Mazzanti, “An- for new peripherally active analgesics for chronic pain timutagenic and Mutagenic Activities of Some Terpenes therapy. 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